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Live-imaging of revertant and therapeutically restored dystrophin in the DmdEGFP-mdx mouse model for Duchenne muscular dystrophy.
Neuropathol Appl Neurobiol. 2020 Jun 23 [Online ahead of print]NA

Abstract

BACKGROUND

Dmdmdx , harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies.

METHODS

We generated a DmdEGFP-mdx reporter allele carrying in cis the mdx-23 mutation and a C-terminal EGFP-tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin-EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo-DNA to skip Dmd exon 23, or after gene editing using AAV-encoded CRISPR/Cas9 for Dmd exon 23 excision.

RESULTS

Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin-EGFP-fluorescence persisted ex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo. Expression of the shorter dystrophin-EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx-23 mutation.

CONCLUSION

Intravital imaging of DmdEGFP-mdx muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo.

Authors+Show Affiliations

Université Paris-Saclay, UVSQ, Inserm, END-ICAP, Versailles, France. Department of Neuropediatrics, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health (BIH), NeuroCure Clinical Research Center, Berlin, Germany.Université Paris-Saclay, UVSQ, Inserm, END-ICAP, Versailles, France.Université Paris-Saclay, UVSQ, Inserm, END-ICAP, Versailles, France.Université Paris-Saclay, UVSQ, Inserm, END-ICAP, Versailles, France.Department of Neuropediatrics, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health (BIH), NeuroCure Clinical Research Center, Berlin, Germany.Department of Neuropediatrics, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health (BIH), NeuroCure Clinical Research Center, Berlin, Germany.Integrare (UMR_S951), Inserm, Généthon, Univ Evry, Université Paris-Saclay, Evry, France.Gustave Roussy, Université Paris-Saclay, Plate-forme Imagerie et Cytométrie., UMS AMMCa., Villejuif, France.Université Paris-Saclay, UVSQ, Inserm, END-ICAP, Versailles, France. LIA BAHN, Centre scientifique de Monaco, Monaco.Université Paris-Saclay, UVSQ, Inserm, END-ICAP, Versailles, France. LIA BAHN, Centre scientifique de Monaco, Monaco.Integrare (UMR_S951), Inserm, Généthon, Univ Evry, Université Paris-Saclay, Evry, France.Gustave Roussy, Université Paris-Saclay, Plate-forme Imagerie et Cytométrie., UMS AMMCa., Villejuif, France.Department of Neuropediatrics, Charité-Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin Institute of Health (BIH), NeuroCure Clinical Research Center, Berlin, Germany.Université Paris-Saclay, UVSQ, Inserm, END-ICAP, Versailles, France. Pediatric Department, University Hospital Raymond Poincaré, Garches, France.

Pub Type(s)

Journal Article

Language

eng

PubMed ID

32573804

Citation

Petkova, M V., et al. "Live-imaging of Revertant and Therapeutically Restored Dystrophin in the DmdEGFP-mdx Mouse Model for Duchenne Muscular Dystrophy." Neuropathology and Applied Neurobiology, 2020.
Petkova MV, Stantzou A, Morin A, et al. Live-imaging of revertant and therapeutically restored dystrophin in the DmdEGFP-mdx mouse model for Duchenne muscular dystrophy. Neuropathol Appl Neurobiol. 2020.
Petkova, M. V., Stantzou, A., Morin, A., Petrova, O., Morales-Gonzalez, S., Seifert, F., Bellec-Dyevre, J., Manoliu, T., Goyenvalle, A., Garcia, L., Richard, I., Laplace-Builhé, C., Schuelke, M., & Amthor, H. (2020). Live-imaging of revertant and therapeutically restored dystrophin in the DmdEGFP-mdx mouse model for Duchenne muscular dystrophy. Neuropathology and Applied Neurobiology. https://doi.org/10.1111/nan.12639
Petkova MV, et al. Live-imaging of Revertant and Therapeutically Restored Dystrophin in the DmdEGFP-mdx Mouse Model for Duchenne Muscular Dystrophy. Neuropathol Appl Neurobiol. 2020 Jun 23; PubMed PMID: 32573804.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Live-imaging of revertant and therapeutically restored dystrophin in the DmdEGFP-mdx mouse model for Duchenne muscular dystrophy. AU - Petkova,M V, AU - Stantzou,A, AU - Morin,A, AU - Petrova,O, AU - Morales-Gonzalez,S, AU - Seifert,F, AU - Bellec-Dyevre,J, AU - Manoliu,T, AU - Goyenvalle,A, AU - Garcia,L, AU - Richard,I, AU - Laplace-Builhé,C, AU - Schuelke,M, AU - Amthor,H, Y1 - 2020/06/23/ PY - 2019/12/27/received PY - 2020/06/03/revised PY - 2020/06/08/accepted PY - 2020/6/24/pubmed PY - 2020/6/24/medline PY - 2020/6/24/entrez KW - CRISPR/Cas9 KW - Duchenne muscular dystrophy KW - dystrophin-EGFP fusion protein KW - mdx reporter mouse model KW - revertant muscle fibre KW - tcDNA JF - Neuropathology and applied neurobiology JO - Neuropathol. Appl. Neurobiol. N2 - BACKGROUND: Dmdmdx , harbouring the c.2983C>T nonsense mutation in Dmd exon 23, is a mouse model for Duchenne muscular dystrophy (DMD), frequently used to test therapies aimed at dystrophin restoration. Current translational research is methodologically hampered by the lack of a reporter mouse model, which would allow direct visualization of dystrophin expression as well as longitudinal in vivo studies. METHODS: We generated a DmdEGFP-mdx reporter allele carrying in cis the mdx-23 mutation and a C-terminal EGFP-tag. This mouse model allows direct visualization of spontaneously and therapeutically restored dystrophin-EGFP fusion protein either after natural fibre reversion, or for example, after splice modulation using tricyclo-DNA to skip Dmd exon 23, or after gene editing using AAV-encoded CRISPR/Cas9 for Dmd exon 23 excision. RESULTS: Intravital microscopy in anaesthetized mice allowed live-imaging of sarcolemmal dystrophin-EGFP fusion protein of revertant fibres as well as following therapeutic restoration. Dystrophin-EGFP-fluorescence persisted ex vivo, allowing live-imaging of revertant and therapeutically restored dystrophin in isolated fibres ex vivo. Expression of the shorter dystrophin-EGFP isoforms Dp71 in the brain, Dp260 in the retina, and Dp116 in the peripheral nerve remained unabated by the mdx-23 mutation. CONCLUSION: Intravital imaging of DmdEGFP-mdx muscle permits novel experimental approaches such as the study of revertant and therapeutically restored dystrophin in vivo and ex vivo. SN - 1365-2990 UR - https://www.unboundmedicine.com/medline/citation/32573804/Live-imaging_of_revertant_and_therapeutically_restored_dystrophin_in_the_DmdEGFP-mdx_mouse_model_for_Duchenne_muscular_dystrophy L2 - https://doi.org/10.1111/nan.12639 DB - PRIME DP - Unbound Medicine ER -
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